Process analytical technology (PAT) is critical for the manufacture of high-quality biologics as it enables continuous, real-time and on-line/at-line monitoring during biomanufacturing processes. The conventional analytical tools currently used have many restrictions to realizing the PAT of current and future biomanufacturing. Here we describe a nanofluidic device for the continuous monitoring of biologics' purity and bioactivity with high sensitivity, resolution and speed. Periodic and angled nanofilter arrays served as the molecular sieve structures to conduct a continuous size-based analysis of biologics. A multiparameter quality monitoring of three separate commercial biologic samples within 50 minutes has been demonstrated, with 20 µl of sample consumption, inclusive of dead volume in the reservoirs. Additionally, a proof-of-concept prototype system, which integrates an on-line sample-preparation system and the nanofluidic device, was demonstrated for at-line monitoring. Thus, the system is ideal for on-site monitoring, and the real-time quality assurance of biologics throughout the biomanufacturing processes.